Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, oomycetes and nematodes. Plants recognize and resist many invading phytopathogens by inducing a rapid defense response. Recognition is often due to the interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant avirulence (Avr) gene product expressed by the invading phytopathogen. R-gene triggered resistance often results in a programmed cell-death, which has been termed the hypersensitive response (HR). The HR is believed to constrain spread of the pathogen.
How R gene products mediate perception of the corresponding Avr proteins is mostly unclear. It has been proposed that phytopathogen Avr products function as ligands, and that plant R gene products function as receptors. In this receptor-ligand model binding of the Avr product to a corresponding R gene product in the plant initiates the chain of events within the plant that produces HR leads to disease resistance. In an alternate model the R protein perceives the action rather than the structure of the Avr protein. In this model the Avr protein is believed to modify a plant target protein (pathogenicity target) in order to promote pathogen virulence. The modification of the pathogenicity protein is detected by the matching R protein and triggers a defense response. Experimental evidence suggests that some R proteins act as Avr receptors while others detect the activity of the Avr protein.
The production of transgenic plants carrying a heterologous gene sequence is now routinely practiced by plant molecular biologists. Methods for incorporating an isolated gene sequence into an expression cassette, producing plant transformation vectors, and transforming many types of plants are well known. Examples of the production of transgenic plants having modified characteristics as a result of the introduction of a heterologous transgene include: U.S. Pat. No. 5,719,046 to Guerineau (production of herbicide resistant plants by introduction of bacterial dihydropteroate synthase gene); U.S Pat. No. 5,231,020 to Jorgensen (modification of flavenoids in plants); U.S. Pat. No. 5,583,021 to Dougherty (production of virus resistant plants); and U.S. Pat. No. 5,767,372 to De Greve and U.S. Pat. No. 5,500,365 to Fischoff (production of insect resistant plants by introducing Bacillus thuringiensis genes).
In conjunction with such techniques, the isolation of plant R genes has similarly permitted the production of plants having enhanced resistance to certain pathogens. Since the cloning of the first R gene, Pto from tomato, which confers resistance to Pseudomonas syringae pv. tomato (Martin et al. (1993) Science 262: 1432-1436), a number of other R genes have been reported (Liu et al. (2007) J. Genet. Genomics 34:765-776). A number of these genes have been used to introduce the encoded resistance characteristic into plant lines that were previously susceptible to the corresponding pathogen. For example, U.S. Pat. No. 5,571,706 describes the introduction of the N gene into tobacco lines that are susceptible to Tobacco Mosaic Virus (TMV) in order to produce TMV-resistant tobacco plants. WO 95/28423 describes the creation of transgenic plants carrying the Rps2 gene from Arabidopsis thaliana, as a means of creating resistance to bacterial pathogens including Pseudomonas syringae, and WO 98/02545 describes the introduction of the Prf gene into plants to obtain broad-spectrum pathogen resistance.
Bacterial spot disease of tomato and pepper, caused by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), can be devastating to commercial production of these crops in areas of the world with high humidity and heavy rainfall. While control of Xcv in commercial agriculture is based largely on the application of pesticides, genetic resistance to bacterial spot disease has been described in both tomato and pepper (Cook and Stall (1963) Phytopathology 53: 1060-1062; Cook and Guevara (1984) Plant Dis. 68: 329-330; Kim and Hartman (1985) Plant Dis. 69: 233-235; Jones and Scott (1986) Plant Dis. 70: 337-339). Of the two hosts, genetic resistance in pepper has been better characterized. Several single loci (Bs1, Bs2, and Bs3) that confer resistance in a “gene-for-gene” manner have been identified (Hibberd et al. (1987) Phytopathology 77: 1304-1307). Moreover, the corresponding avirulence genes (avrBs1, avrBs2, and avrBs3) have been cloned from Xcv (Swanson et al. (1988) Mol. Plant-Microbe Interact. 1:5-9; Minsavage et al. (1990) Mol. Plant-Microbe Interact. 3: 41-47). Genetic and molecular characterization of these avirulence genes has provided a great deal of information concerning the interaction between Xcv and pepper (Kearney et al. (1988) Nature 332: 541-543; Kearney and Staskawicz (1990) Nature 346: 385-386; Herbers et al. (1992) Nature 356: 172-174; Van der Ackerveken et al. (1992) Plant J. 2: 359-366). More recently, the Bs3 gene of pepper has been isolated and sequenced (U.S. Pat. No. 6,262,343)
Xcv employs a type III secretion (T3S) system to inject an arsenal of about 20 effector proteins into the host cytoplasm that collectively promote virulence (Thieme et al. (2005) J. Bacteriol. 187:7254). R protein mediated defense in response to Xcv effector proteins is typically accompanied by a programmed cell death response referred to as the HR. AvrBs3 is one Avr protein that R proteins recognize and is a member of large family (>100 sequenced members) of highly related bacterial effector proteins that are present in various Xanthomonas and Ralstonia solanacearum strains (Schornack et al. (2006) J. Plant Physiol. 163:256). Due to their structural relatedness to eukaryotic transcription factors AvrBs3-like proteins are also referred to as TAL (transcription activator like) effectors. The most characteristic feature of TAL effectors is the central repeat domain that consists of a variable number (1.5-28.5) of tandem-arranged, almost identical 34/35-(Xanthomonas/Ralstonia) repeat units. Analysis of AvrBs3 from Xcv has shown that the repeat domain mediates specific binding to a promoter element that has been termed “upa box” (Kay et al. (2007) Science 318:648-651). The full length AvrBs3 protein not only binds to promoters with a upa box but also transcriptionally activates these promoters. In pepper genotypes that are susceptible to Xcv, AvrBs3 binds to and activates the promoter of the upa20 gene, which causes cell hypertrophy (Kay et al. (2007) Science 318:648-651). In pepper plants that contain the Bs3 resistance gene, AvrBs3 triggers a cell death response (i.e., HR) that restricts pathogen growth. Molecular analysis revealed that the Bs3 promoter contains, like the upa20 promoter, a upa box. AvrBs3 binds to and transcriptionally activates the pepper Bs3 promoter thereby triggering a defense reaction (Römer et al. (2007) Science 318:645-648). Thus the Bs3 promoter represents a DNA-based decoy receptor. The AvrBs3-deletion derivative AvrBs3Δrep16 (lacks repeat units 11-14) does not activate the Bs3 promoter but its allelic variant Bs3-E (Römer et al. (2007) Science 318:645-648). Intriguingly the Bs3 and Bs3-E promoter differ in their upa boxes (herein referred to as “upaAvrBs3” and “upaAvrBs3Δrep16” boxes, respectively). Thus recognition specificity of TAL effectors is determined by a) the sum of the repeat units of a given TAL effector and b) the upa box of a given host promoter.
The TAL effector AvrXa27 from the bacterial rice pathogen Xanthomonas oryzae pv. oryzae (Xoo) activates the promoter of the matching rice R gene, Xa27 (Gu et al. (2005) Nature 435:1122-1125). Thus, the R genes Bs3 and Xa27 are both transcriptionally activated by their matching TAL effectors and thus are identical in their mechanisms of activation. However, the predicted Bs3 and Xa27 proteins share neither sequence homology to each other nor to the classical NB-LRR type R proteins. Nevertheless, it seems likely that AvrXa27- and AvrBs3-mediated activation of host promoters are mechanistically similar. To date, no report has yet appeared which provides evidence demonstrating that AvrXa27 binds to the Xa27 promoter and that the Xa27 promoter contains a upa box to which AvrXa27 binds.